Project Summary/Abstract The proposal outlines an integrated research and career development plan for David Wu, MD PhD, to com- plete postdoctoral training in the laboratories of Gokhan Mutlu, MD, and Yun Fang, PhD and transition to an independent academic position by establishing a research program in vascular pathophysiology with a NIH mentored career award (K99/R00). The PI has recently completed an NIH F32 fellowship (F32 HL134288) and is trained in the fields of metabolism, molecular biology, biophysics, optics, bioengineering, and vascular biolo- gy. During the 2-year mentored period (K99), the PI will receive additional academic guidance from the men- tors and the advisory committee at the University of Chicago. The career development plan is designed to equip the PI with the necessary knowledge and skills in biomedical research for a successful transition as an independent academician, leading to an R01 as the R00 phase of the work progresses. The overall research goal is to determine the role of endothelial metabolism in mediating endothelial phenotypes in relation to the initiation and propagation of acute lung injury. The role of endothelial metabolism in pro-inflammatory hemody- namical flow types is poorly understood. Preliminary studies conducted by the PI during his post-doctoral F32 phase demonstrated that low shear stress or disturbed flow hemodynamics causes endothelial cells to in- crease glycolytic metabolism, compared to unidirectional flow. Increasing this glycolysis is necessary for in- creased endothelial inflammation (eLife, 2017). The PI furthermore showed that in acute lung injury, endotheli- al cells are unable to sense hemodynamic flow, and have a phenotype that is reminiscent of disturbed flow hemodynamics (AJRCCM, 2017). This K99/R00 research proposal tests the overall hypothesis that endothelial cell dysfunction causes a change in endothelial metabolism that is critical to the initiation and progression of acute lung injury. Aim 1 will test the hypothesis that the cytoskeleton is critical for initiating localized glycolysis that powers vascular barrier breakdown by RhoA mediated release of aldolase A, a critical enzyme in the gly- colytic pathway. Aim 2 will test the hypothesis that down-regulation of oxidative phosphorylation as a conse- quence of endothelial cell dysfunction causes mitochondrial reverse electron transport, which is critical for re- active oxygen species production and upregulation of glycolysis. Aim 3 will test the hypothesis that modulating endothelial metabolism can reduce lung injury in mouse models. The goal will be achieved by integrating single cell microscopy and molecular analysis in vitro and in vivo systems, leading to a mechanistic understanding of how metabolism influences injury propagation, and perhaps uncover therapeutic targets. Attainment of this proposed career award will accelerate the transition for David Wu to an independent physician-scientist and lead to acquisition of competitive R01 funding.